Adventitial Vasa Vasorum Arteriosclerosis in Abdominal

Aortic Aneurysm
Hiroki Tanaka1,2., Nobuhiro Zaima2,4., Takeshi Sasaki3, Takahiro Hayasaka2, Naoko Goto-Inoue2,
Kenji Onoue2, Koji Ikegami2, Yoshifumi Morita1,2, Naoto Yamamoto1, Yuuki Mano1, Masaki Sano1,
Takaaki Saito1, Kohji Sato3, Hiroyuki Konno1, Mitsutoshi Setou2*, Naoki Unno1*
1 Second Department of Surgery, Hamamatsu University School of Medicine, Hamamatsu, Japan, 2 Department of Cell Biology and Anatomy, Hamamatsu University
School of Medicine, Hamamatsu, Japan, 3 Department of Anatomy and Neuroscience, Hamamatsu University School of Medicine, Hamamatsu, Japan, 4 Department of
Applied Biological Chemistry, Kinki University, Higashiosaka City, Japan

Abstract
Abdominal aortic aneurysm (AAA) is a common disease among elderly individuals. However, the precise pathophysiology of
AAA remains unknown. In AAA, an intraluminal thrombus prevents luminal perfusion of oxygen, allowing only the
adventitial vaso vasorum (VV) to deliver oxygen and nutrients to the aortic wall. In this study, we examined changes in the
adventitial VV wall in AAA to clarify the histopathological mechanisms underlying AAA. We found marked intimal
hyperplasia of the adventitial VV in the AAA sac; further, immunohistological studies revealed proliferation of smooth
muscle cells, which caused luminal stenosis of the VV. We also found decreased HemeB signals in the aortic wall of the sac
as compared with those in the aortic wall of the neck region in AAA. The stenosis of adventitial VV in the AAA sac and the
malperfusion of the aortic wall observed in the present study are new aspects of AAA pathology that are expected to
enhance our understanding of this disease.
Citation: Tanaka H, Zaima N, Sasaki T, Hayasaka T, Goto-Inoue N, et al. (2013) Adventitial Vasa Vasorum Arteriosclerosis in Abdominal Aortic Aneurysm. PLoS
ONE 8(2): e57398. doi:10.1371/journal.pone.0057398
Editor: Elena Aikawa, Brigham and Womens Hospital, Harvard Medical School, United States of America
Received September 7, 2012; Accepted January 21, 2013; Published February 27, 2013
Copyright: 2013 Tanaka et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the following grants: a Grant-in-Aid for Scientific Research (C) (22590522) (to NZ); Project of Hamamatsu University
Research Promotion (to NU); a Grant-in-Aid for SENTAN from the Japan Science and Technology Agency; and Tokutei Lipid Machinery and Young Scientists S
(2067004) (to MS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: unno@hama-med.ac.jp (NU); setou@hama-med.ac.jp (MS)
. These authors contributed equally to this work.

morphological analysis of VV, in fresh surgical samplesfrom

patients undergoingopen repair of AAA. We further assessed the
distribution of lipid molecules in the VV wall using matrix-assisted
laser desorption/ionization imaging mass spectrometry (MALDIIMS), to profile discrete cellular regions and obtain region-specific
images, providing information on the relative abundance and
spatial distribution of proteins, peptides, lipids, [6] and drugs [7].

Introduction
Abdominal aortic aneurysm (AAA) involves the progressive
dilatation of the abdominal aorta as a consequence of degeneration. Currently,surgical repair is the only available method
of treatment [1] since lack of knowledge regarding the pathogenesis of AAA has hindered the development of suitable medical
treatments.
One of the proposed mechanisms of AAA development/rupture
is hypoxia-mediated weakening of the wall [2,3]. The aortic wall is
normally maintained by direct perfusion from the vessel lumen or
perfusion via the adventitial vaso vasorum (VV). The presence of
an intraluminal thrombus (ILT) is thought to prevent the luminal
perfusion of oxygen to the aortic wall, and this could cause tissue
hypoxia. The role played byVV in the perfusion of the aortic wall
in AAA remains unknown. The VV delivers nutrients and oxygen
to the arterial wall and removes waste products produced in the
wall [4]. Interestingly, the distribution of the VV in the abdominal
aorta is known to be reduced in the infrarenal abdominal aorta as
compared with that in the thoracic aorta [5].
We hypothesized that damage to the VV may be associated
with disturbances in the delivery of nutrients and oxygen to the
aortic wall, and thus may play an important role in the
pathogenesis of AAA. In this study, we therefore examined the
changes occurring in the adventitial VV in patients with AAA
PLOS ONE | www.plosone.org

Materials and Methods

Sample Collection
All procedures used in this study were approved by the Ethics
Committee of Clinical Research of the Hamamatsu University
School of Medicine, and with written consent was obtained
from each patient. We enrolled 30 patients who underwent
elective open surgery for repair of infrarenal AAAs at the
Division of Vascular Surgery, Hamamatsu University School of
Medicine, between April 2008 and April 2011. Aortic tissue
samples were dissected during surgery abased on preoperative
three-dimensional multi-detector computed tomography (3DMDCT) imaging of the AAA being excised from the patient
(Fig. 1AC). Longitudinal tissue strips were selected from the
infrarenal aortic neck (non-dilated normal aorta). Similarstrips
extendinginto the region of maximal aneurysmal dilation were
also obtained (Fig. 1D and 1E).
1

February 2013 | Volume 8 | Issue 2 | e57398

Malperfusion in AAA Wall

Figure 1. Sample harvesting during open repair of AAA. (A) Preoperative contrast-enhanced 3D-MDCT images of a patient (Table 1) with an
AAA. A, anterior; D, dorsal; L, left; P, posterior; R, right; V, ventral. Scale bar = 2.0 cm. (B) Intraoperative view. Scale bar = 2.0 cm. (C) Schema of the AAA
tissue. (D) Isolated tissue. Scale bar = 1.0 cm. (E) Frozen tissue before cross sectioning. Scale bar = 1.0 cm.
doi:10.1371/journal.pone.0057398.g001

Fine Chemical Co., Ltd., Tokyo, Japan), goat anti-cathepsin S

(1:100; Santa Cruz Biotechnology, Inc.), and mouse anti- hypoxic
inducible factor-1a (HIF-1a) (1:200; Novus Biological, LLC, CO,
USA).
The lumen and medial areas of the VV were measured in each
section. The luminal area was defined as the area enclosed by the
intima, while the intima-medial area was defined as the area
enclosed between the external elastic laminae and the lumen. The
average values of these areas in the adventitial VV from the neck
and sac of AAA samples were compared. These areas were also
measured in the adventitial VV of the control autopsy samples,
specifically from the mid-portion of the infrarenal aorta.

Eleven aorta samples obtained at autopsy were used as controls.

The mid-portion of the abdominal aorta between the renal artery
and the bifurcation was resected and collected from routine
autopsies in the Department of Pathology, Hamamatsu University
Hospital.

Immunofluorescence Staining
Three tissue sections (8-mm thick) from the neck and sac of each
AAA sample were obtained. The tissue sections were fixed with
4% paraformaldehyde in phosphate-buffered saline (pH 7.4) for
10 min at room temperature. The histological results from the
VVs were assessed after staining using the following: rabbit antialpha smooth muscle actin (1:100; Thermo Scientific, Waltham,
MA, USA) or rabbit anti-Ki-67 (Ki-67) (1:100; Abcam, Cambridge, MA, USA), mouse anti-Calprotectin-Monocyte/Macrophage (1:100; Thermo Scientific), mouse anti-CD3e (1:100;
Thermo Scientific), goat anti-CD20 (1:100; Santa Cruz Biotechnology, Inc., Santa Clara, CA, USA), rabbit anti-MMP-2
(1:100; Thermo Scientific), mouse anti-MMP-9 (1:100; Daiichi

PLOS ONE | www.plosone.org

Imaging Mass Spectrometry

Matrix-assisted laser desorption (MALDI)/ionization mass
spectrometry (IMS) was performed on the freshly harvested
specimens from AAA patients. Three sections in the neck and
sac of each AAA sample were obtained. Samples were prepared as
described previously [6]. MALDI/IMS was performed using

February 2013 | Volume 8 | Issue 2 | e57398

Malperfusion in AAA Wall

adventitia was associated with the proliferation of the intimamedial smooth muscle cells (SMCs) (Fig. 3A). Ki-67 staining was
significantly more positive in the VV SMCs in the AAA sac than
that observed in consecutive sections of the control and AAA neck
VV (Fig. 3B). No atherosclerotic plaques were observed in the VV
lumens in any of the specimens. Further, HIF-1a immunoreactivity was positive in the SMCs of the VV, suggesting that the VV
wall was hypoxic (Fig. 3C). HIF-1a stained positive in all the layers
of the AAA sac wall, which corresponded to the areas with weak
Heme B signals (which is a specific marker for blood stock and
may reflect the level of tissue blood flow) on MALDI-IMS (Fig. 4A
and 4B). MALDI-IMS revealed that the relative intensity of Heme
B (m/z 616) in the sac was significantly lower than that in the neck
in both the intima and media as well as in the adventitia (Fig. 5).
PC(16:0/18:1), the internal standard molecule, was detected
ubiquitously both in the AAA neck and sac. Further, both
MMP-2 and MMP-9 were strongly positive in the media and
adventitia of the AAA sac wall, while cathepsin stained positively
only in the adventitial side of the AAA sac wall. Co-immunostaining of macrophages/T-cells and HIF-1a identified that HIF-1a
immunoreactivity was also positive in the macrophages in the
AAA sac media and adventitia but negative in T-cells (Fig. 4C and
4D). MALDI-IMS further revealed that proinflammatory lipid
molecules such as lyso-PC (LPC) (1-acyl 16:0) and PC(16:0/20:4)
were distributed in all the layers of the AAA sac; however,
significantly greater accumulation of these molecules was observed
in the adventitial side. In contrast, the distribution of cholesterol
ester (CE) was not significantly different between the AAA neck
and sac arterial walls.
MALDI-IMS also showed characteristic localization of lyso-PC
(LPC) (1-acyl 16:0) and PC diacyl (16:0/20:4) in the sac VV as
compared with the neck VV (Fig. 6A and 6B). CE(18:1) was not
detected either in the neck or sac VV, indicating no atherosclerotic
lesions in the VV. PC diacyl (16:0/18:1), the internal standard
molecule, was detected ubiquitously in both the neck and sac VV
tissues.

a time of flight (TOF) instrument (Ultraflex II, Bruker Daltonics

Inc., Billerica, MA, USA) equipped with a 355-nm Nd:YAG laser
at a repetition rate of 200 Hz. Data were acquired with a step size
of 100 mm in the positive ion mode (reflector mode). A total of
500 mL of 2,5-dihydroxybenzoic acid (2, 5-DHB) solution in
methanol/water (7/3, v/v) was used as the matrix. After analysis
by IMS, the sections were subjected to hematoxylin-eosin (HE)
staining.

Identification of Biomolecules
To identify the peaks, MS/MS was performed using a MALDIquadrupole ion trap (QIT)-TOF mass spectrometer (AXIMAQIT; Shimadzu, Kyoto, Japan) [8]. The specific fragment patterns
of HemeB, phosphatidylcholines (PCs), and cholesteryl esters (CEs)
were annotated according to previous reports [8,9].

Statistical Analysis
The results were analyzed using StatView software (version 5.0;
SAS Institute, Cary, NC, USA). All data were expressed as mean
values 6 SD. Statistical analysis was performed using analysis of
variance for comparisons among the 3 groups (control, and neck
and sac regions). Post-hoc comparison was performed using
Tukeys test. Comparisons between the neck and the sac regions
were evaluated using the paired t test.

Results
The clinical characteristics of the AAA patients are shown in
Table 1. Eleven sex- and age-matched autopsy cases served as
controls: their ages ranged from 54 to 89 years (mean age
71.269.7 years, all men). Of the control cases, 7 patients had died
of cancer; 2, of renal failure; 1, of cerebral stroke; and 1, of cardiac
infarction.
Histopathological analysis revealed marked intimal hyperplasia
in the adventitial VV ofthe AAA sac with a compromised luminal
area as compared with the VV in the non-dilated neck adventitia
(Fig. 2A and 2B). Moreover, these changes in the VV were not
observed in the control specimens. Due to the intimal hyperplasia,
the luminal area of the VV in the sac adventitia was significantly
smaller than that in the non-dilated neck adventitia or in the
control specimens (Fig. 2C). Stenosis of the VV in the sac

Discussion
The present study revealed significant stenosis of the adventitial
VV in the arterial wall of the AAA sac, resulting from the
proliferation of SMCs, which stained positive for HIF-1a. HIF-1a
was also positive in the intima and medial layers, indicating
hypoxia of the arterial wall. We further observed a decreased
HemeB level in the sac wall, suggesting low perfusion of the sac.
HemeB is the most abundant heme. It is synthesized and
distributed in erythroid cells and hepatocytes, indicating that
Heme B is a more specific marker for blood stock than formic acid
or iron that can accurately reflect the tissue blood flow status [8]
[10]. Therefore, in the present study, we assayed HemeB levels in
order to examine the tissue blood flow.
The aortic wall is normally maintained by direct perfusion from
the vessel lumen or perfusion via the adventitial VV. Inmost cases
of AAA, large ILT occurs concurrently; therefore, oxygen delivery
by direct perfusion from the lumen to the sac wall is expected to
becompromised [11]. Among the 30 cases included in this study,
23 (.75%) cases showed the presence of a massive ILT, while no
ILT was noted in 7 cases. We compared the two groups with/
without ILT to assess whether the presence of ILT influenced
adventitial VV arteriosclerosis or the expression of other molecules
such as HIF-1a, Heme B, MMP-9, etc. However, no differences
were observed between these two groups, indicating that changes
in adventitial VV occurred irrespective of the presence of ILT
(data not shown). Therefore, we consider that adventitial VV may

Table 1. Demographic and clinical data of the AAA patients.

Sex (n) (male/female)

26/4

Age

70.269.0

Height (m)

1.660.1

Weight (kg)

58.0610.8

BMI (kg/m2)

21.962.4

Aortic diameter (mm)

Neck
Sac
Serum TC (mg/dL)

21.362.2
54.6612.2
192.3630.8

HbA1c (%)

5.560.7

CRP (,0.3) (mg/dL)

1.062.2

Ever smoked (n)

30

Values are presented as mean 6 SD unless stated otherwise.

Normal ranges: TC 130240 mg/dL; HbA1c 4.35.8%; CRP 4.35.8 mg/dL.
AAA, abdominal aortic aneurysm; BMI, body mass index; TC, total cholesterol.
doi:10.1371/journal.pone.0057398.t001

PLOS ONE | www.plosone.org

February 2013 | Volume 8 | Issue 2 | e57398

Malperfusion in AAA Wall

Figure 2. Adventitial vasa vasorum (VV) and its luminal stenosis. (A) Representative adventitial VV with Elastica Van Gieson (EVG) staining.
Patent VV in the control and abdominal aortic aneurysm (AAA) neck, andstenotic VV in the AAA sac. Scale bar = 200 mm. (B) Comparison of the
luminal and intimamedial areas among the control, neck, and sac adventitial VV. The luminal area was defined as the area enclosed by the intima,
while the intimamedial area was defined as the area enclosed between the external elastic laminae and the lumen. Scale bar = 100 mm. (C) Data
were obtained from 7 patients and 5 autopsied cases (controls). *P,0.001 indicates a statistically significant difference.
doi:10.1371/journal.pone.0057398.g002

play an independent role in the perfusion/oxygenation of the

aortic wall, with VV stenosis contributing to the ischemia of the
aortic wall itself. Given that the number of VV in the abdominal
aorta is much less than that in the thoracic aorta, luminal stenosis
of the sac VV could exacerbate tissue hypoxia in the abdominal
aorta.
Pathologically, most aneurysms are characterized by upregulated proteolytic pathways within the medial layer of the arterial
wall along with oxidative stress, adventitial inflammation, and loss
of wall matrix [11,12,13]. Increased matrix metalloproteinase
(MMP) activity has been implicated in aneurysm formation
through elastin destruction and collagen degradation [14]. The

concept that hypoxia can induce inflammation has gained general

acceptance. The expression of HIF-1a is an adaptive response by
the body against hypoxia, which in turn affects various inflammatory mediators [15]. MMP-2 one of these mediators, is
activated under hypoxic conditions by a signaling cascade
involving HIF-1a [16]. In our study, HIF-1a stained positive in
both SMCs and macrophages in the AAA sac wall, indicating the
occurrence of hypoxic conditions. Recently, Wan et al. reported
that the accumulation of HIF-1a might be involved in the
increased production of MMP-2 and MMP-9 in human monocytes [17]. Therefore, hypoxia (or HIF-1a) may accelerate MMP
production from macrophages. Thus, intimal hyperplasia in the

Figure 3. Immunohistological study of adventitial vasa vasorum (VV). (A) Immunostaining of SMCs in VV expressing alpha smooth muscle
actin. Scale bar = 100 mm. (B) Immunostaining of cell proliferation marker (Ki-67) in VV. Scale bar = 20 mm. (C) Immunostaining of hypoxic inducible
factor-1a (HIF-1a) in VV. Scale bar = 20 mm.
doi:10.1371/journal.pone.0057398.g003

PLOS ONE | www.plosone.org

February 2013 | Volume 8 | Issue 2 | e57398

Malperfusion in AAA Wall

Figure 4. Distribution of lipid molecules and HemeB. (A) Elastica Van Gieson (EVG) staining and immunostaining of HIF-1a, MMP-2, MMP-9,
and Cathepsin S in the aortic wall. Comparison of the distribution of these molecules in the aortic wall between the neck and sac regions. Brown color
indicates the positive signals in all immunostaining pictures. Scale bar = 50 mm (from left to right). (B) Comparison of the distribution of Heme B,
phosphatidylcholine (PC) (16:0/18:1), lyso-PC (LPC) (1-acyl 16:0), PC(16:0/20:4), and cholesteryl ester (CE) (18:1), in the aortic wall between the neck
and sac regions, as analyzed by matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS). Scale bar = 100 mm. (C) HIF-1a,
macrophages, and T cells in the AAA sac. Scale bar = 200 mm. (D) Immunofluorescence analysis of HIF-1a and macrophages in the AAA sac. Scale
bar = 5 mm.
doi:10.1371/journal.pone.0057398.g004

VV and the subsequent ischemia/hypoxia may be the major

pathogenic mechanism involved in the development of AAA.
However, the present study could not clarify whether VV stenosis
occurred before degeneration and expansion of the aortic wall or
resulted from changes occurring during the disease process.
Further studies are required to clarify this issue.
VV stenosis occurs not because of atherosclerotic plaque
formation but because of intimal hyperplasia. To the best of our

PLOS ONE | www.plosone.org

knowledge, no epidemiological study has investigated the risk

factors for VV stenosis. However, considering that VV are
essentially endarteries, it is possible that aging, compression due to
hypertension, and smoking could affect VV circulation; these
factors are all known as important risk factors for AAA.
Interestingly, the abdominal aorta appears to be particularly
susceptible to the effects of smoking [18,19].

February 2013 | Volume 8 | Issue 2 | e57398

Malperfusion in AAA Wall

Furthermore, inflammation may provoke release of growth factors

and cytokines such as PDGF, FGF, IGF, and IL-1, which further
induce intimal hyperplasia [25,26,27,28].Thus, reduced VV blood
flow, and the subsequent tissue hypoxia and inflammation may
promote the proliferation of SMCs and accelerate the progression
of luminal stenosis of VV, which in turn exacerbates hypoxia in
the aortic wall.
To gain further insight into the pathogenesis of intimal
hyperplasia in the VV, we analyzed the distribution of lipid
molecules using MALDI-IMS. At present, MALDI-IMS is the
only method available for the detailed assessment of lipid
molecules in tissues. This method distinguishes between different
lipid molecular species by simultaneously determining the
differences in the mass/charge ratios (m/z). MALDI-IMS revealed
abnormal accumulation of the lipid molecules LPC(1-acyl 16:0)
and PC(16:0/20:4) in the AAA sacs medial and adventitial layers,
where Heme B distribution was also remarkably reduced. Both
LPC(1-acyl 16:0) and PC(16:0/20:4) were also observed to be
accumulated in VV tissues in the AAA sac wall. Since MALDIIMS can only be performed using fresh samples, we could not
evaluate the control samples using this method. However,
abnormal accumulation of the lipid molecules was not observed
in the neck VV tissue but was observed in the sac VV tissue. We
have previously reported the accumulation of both PC(16:0/20:4)
and LPC(1-acyl 16:0) in intimal hyperplasia of the femoral artery
in patients with peripheral artery occlusive disease. [9] Although
the range of biochemical effects of PC (16:0/20:4) is largely
unknown, elevated levels of the molecule have been reported in
mammalian tissues with chronic inflammation [29,30].
The PC class of lipids is a major component of most
intracellular membranes. It is composed of a choline head group,
a phosphoglycerol backbone, and 2 acyl chains of various
combinations, which generate various PC species. PC(16:0/20:4)
is an arachidonic acid-containing PC, which is degraded to
arachidonic acid and LPC(1-acyl 16:0) by phospholipase A2 [31].
Arachidonic acid induces proinflammatory signaling and interacts
with substances in the vascular wall leading to the accumulation of
foam cells and development of intimal hyperplasia [32,33]. In

Figure 5. Comparison of HemeB signals. Comparisons of the

relative intensity of Heme B/PC(16:0/18:1) in both intimamedial and
adventitial regions in the AAA sac wall as compared with those in the
neck wall. *P,0.001 indicates a statistically significant difference.
doi:10.1371/journal.pone.0057398.g005

Anatomically, VV originate from various arteries. The VV in

the ascending aorta are derived from the coronary arteries and
brachiocepharic artery. The VV in the descending thoracic aorta
are derived from the intercostal arteries, while the VV in the
abdominal aorta are derived from the lumbar and mesenteric
arteries [20]. In AAA, the presence of an ILT frequently occludes
the orifices of lumbar arteries or the inferior mesenteric artery,
which may decrease the blood flow to the VV. The reduced blood
flow in the VV could then cause proliferation of SMCs due to low
wall shear stress [21,22]. Hypoxia may also stimulate the
proliferation of vascular SMCs via an HIF-1a-mediated pathway
[23]. Indeed, in the sac adventitial VV SMCs, Ki-67 staining was
significantly more positive than that in neck and control VV
SMCs, suggesting that sac VV SMCs might have a greater
proliferative ability than control and AAA neck VV SMCs [24].

Figure 6. Distribution of lipid molecules in VV in high resolution. (A) MALDI-IMS. Comparison of the distribution of lipid molecules,
phosphatidylcholine (PC) (16:0/18:1), lyso-PC (LPC) (1-acyl 16:0), PC(16:0/20:4), and cholesteryl ester (CE) (18:1), in the VV wall between the neck and
sac regions analyzed by matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS). Scale bar = 20 mm. (B) Comparisons of
the relative intensity of LPC(1-acyl 16:0)/PC(16:0/18:1) and PC(16:0/20:4)/PC(16:0/18:1) in the VV wall between the neck and sac regions analyzed by
MALDI-IMS. *P,0.001 indicates a statistically significant difference.
doi:10.1371/journal.pone.0057398.g006

PLOS ONE | www.plosone.org

February 2013 | Volume 8 | Issue 2 | e57398

Malperfusion in AAA Wall

animal models of aneurysm, leukotrienes which are produced from

arachidonic acid have been reported to weaken the adventitia of
the aorta [34]. Therefore, accumulation of arachidonic acidcontaining PC in the VV wall may be implicated in an ongoing
arachidonic acid-related inflammatory cascade. Moreover, the
other molecule detected by MALDI-IMS, i.e., LPC, is known to
exert a variety of biological activities, such as activating Tlymphocytes and enhancing adhesion molecule expression in
endothelial cells [35]. It also plays an important role in the
mitogenic activity of oxidized low-density cholesterol in monocytederived macrophages [36]. Therefore, the accumulation of
PC(16:0/20:4) and LPC(1-acyl 16:0) in the AAA sac VV could
be associated with tissue inflammation, eventually leading to VV
intimal hyperplasia.

Immunohistological and MALDI-IMS findings indicated that the

aortic wall of the AAA sac was ischemic and hypoxic. Our findings
validate the role of changes in the VV of the AAA sac and the
subsequent hypoxia/ischemia of the aortic wall in the pathogenesis
of AAA.

Acknowledgments
We thank the staff of the Second Department of Surgery of the
Hamamatsu University School of Medicine. We are grateful to Y.
Sugiyama, S. Kamei and Y. Kaneko for their assistance. Parts of these
results have been published in several meetings, such as the meeting of the
International Mass Spectrometry Society.

Author Contributions
Conceived and designed the experiments: HT NZ M. Setou NU.
Performed the experiments: HT NZ TS. Analyzed the data: HT NZ TS
TH NG-I KO KI YM NY YM M. Sano M. Setou TS KS HK MT NU.
Contributed reagents/materials/analysis tools: NY TH YM M. Sano TS
NU. Wrote the paper: HT NZ M. Setou NU.

Conclusion
In this study, we demonstrated stenosis of the adventitial VV in
the AAA sac with intimal hyperplasia of the VV. Accumulation of
abnormal lipid molecules in the sac VV tissues was also observed.

References
1. Lindsay ME, Dietz HC (2011) Lessons on the pathogenesis of aneurysm from
heritable conditions. Nature 19473: 308316.
2. Vorp DA, Lee PC, Wang DH, Makaroun MS, Nemoto EM, et al. (2001)
Association of intraluminal thrombus in abdominal aortic aneurysm with local
hypoxia and wall weakening. J Vasc Surg 34: 291299.
3. Choke E, Cockerill GW, Dawson J, Chung YL, Griffiths J, et al. (2006) Hypoxia
at the site of abdominal aortic aneurysm rupture is not associated with increased
lactate. Ann N Y Acad Sci 1085: 306310.
4. Ritman EL, Lerman A (2007) The dynamic vasa vasorum. Cardiovasc Res 75:
649658.
5. Wolinsky H, Glagov S (1969) Comparison of abdominal and thoracic aortic
medial structure in mammals. Deviation of man from the usual pattern. Circ Res
25: 677686.
6. Tanaka H, Zaima N, Yamamoto N, Sagara D, Suzuki M, et al. (2010) Imaging
mass spectrometry reveals unique lipid distribution in primary varicose veins.
Eur J Vasc Endovasc Surg 40: 657663.
7. Stoeckli M, Chaurand P, Hallahan DE, Caprioli RM (2001) Imaging mass
spectrometry: a new technology for the analysis of protein expression in
mammalian tissues. Nat Med 7: 493496.
8. Shimma S, Sugiura Y, Hayasaka T, Zaima N, Matsumoto M, et al. (2008) Mass
imaging and identification of biomolecules with MALDI-QIT-TOF-based
system. Anal Chem 80: 878885.
9. Tanaka H, Zaima N, Yamamoto N, Suzuki M, Mano Y, et al. (2011)
Distribution of phospholipid molecular species in autogenous access grafts for
hemodialysis analyzed using imaging mass spectrometry. Anal Bioanal Chem
400: 18731880.
10. Ponka P (1997) Tissue-specific regulation of iron metabolism and heme
synthesis: distinct control mechanisms in erythroid cells. Blood 89: 125.
11. Sakalihasan N, Limet R, Defawe OD (2005) Abdominal aortic aneurysm.
Lancet 365: 15771589.
12. Michel JB, Thaunat O, Houard X, Meilhac O, Caligiuri G, et al. (2007)
Topological determinants and consequences of adventitial responses to arterial
wall injury. Arterioscler Thromb Vasc Biol 27: 12591268.
13. Ramos-Mozo P, Madrigal-Matute J, Martinez-Pinna R, Blanco-Colio LM,
Lopez JA, et al. (2011) Proteomic analysis of polymorphonuclear neutrophils
identifies catalase as a novel biomarker of abdominal aortic aneurysm: potential
implication of oxidative stress in abdominal aortic aneurysm progression.
Arterioscler Thromb Vasc Biol 31: 30113019.
14. Norman PE, Powell JT (2010) Site specificity of aneurysmal disease. Circulation
121: 560568.
15. Eltzschig HK, Carmeliet P (2011) Hypoxia and inflammation. New Engl J Med
364: 656665.
16. Erdozain OJ, Pegrum S, Winrow VR, Horrocks M, Stevens CR (2011) Hypoxia
in abdominal aortic aneurysm supports a role for HIF-1a and Ets-1 as drivers of
matrix metalloproteinase upregulation in human aortic smooth muscle cells.
J Vasc Res 48: 163170.
17. Wan R, Mo Y, Chien S, Li Y, Li Y, et al. (2011) The role of hypoxia inducible
factor-1a in the increased MMP-2 and MMP-9 production by human
monocytes exposed to nickel nanoparticles. Nanotoxicology 5: 568582.
18. VanderLaan PA, Reardon CA, Getz GS (2004) Site specificity of atherosclerosis:
site-selective responses to atherosclerotic modulators. Arterioscler Thromb Vasc
Biol 24: 1222.
19. Kent KC, Zwolak RM, Egorova NN, Riles TS, Manganaro A, et al. (2010)
Analysis of risk factors for abdominal aortic aneurysm in a cohort of more than 3
million individuals. J Vasc Surg 52: 539548.

PLOS ONE | www.plosone.org

20. Clarke JA (1965) An x-ray microscopic study of the postnatal development of the
vasa vasorum in the human aorta. J Anat 99: 877889.
21. Fan L, Karino T (2010) Effect of a disturbed flow on proliferation of the cells of
a hybrid vascular graft. Biorheolgy 47: 3138.
22. Chiu JJ, Chien S (2011) Effects of disturbed flow on vascular endothelium:
pathophysiological basis and clinical perspectives. Physiol Rev 91: 327387.
23. Schultz K, Fanburg BL, Beasley D (2006) Hypoxia and hypoxia-inducible
factor-1alpha promote growth factor-induced proliferation of human vascular
smooth muscle cells. Am J Physiol Heart Circ Physiol 290: H25282534.
24. Ikesue M, Matsui Y, Ohta D, Danzaki K, Ito K, et al. (2011) Syndecan-4
deficiency limits neointimal formation after vascular injury by regulating
vascular smooth muscle cell proliferation and vascular progenitor cell
mobilization. Arterioscler Thromb Vasc Biol 31: 10661074.
25. Doran AC, Meller N, McNamara CA (2008) Role of smooth muscle cells in the
initiation and early progression of atherosclerosis. Arterioscler Thromb Vasc
Biol 28: 812819.
26. Brog iE, Winkles JA, Underwood R, Clinton SK, Alberts GF, et al. (1993)
Distinct patterns of expression of fibroblast growth factors and their receptors in
human atheroma and nonatherosclerotic arteries. Association of acidic FGF with
plaque microvessels and macrophages. J Clin Invest 92: 24082418.
27. Shai SY, Sukhanov S, Higashi Y, Vaughn C, Kelly J, et al. (2010) Smooth
muscle cell-specific insulin-like growth factor-1 overexpression in Apoe2/2
mice does not alter atherosclerotic plaque burden but increases features of
plaque stability. Arterioscler Thromb Vasc Biol 30: 19161924.
28. Isoda K, Shiigai M, Ishigami N, Matsuki T, Horai R, et al. (2003) Deficiency of
interleukin-1 receptor antagonist promotes neointimal formation after injury.
Circulation 108: 516518.
29. Koizumi S, Yamamoto S, Hayasaka T, Konishi Y, Yamaguchi-Okada M, et al.
(2010) Imaging mass spectrometry revealed the production of lyso-phosphatidylcholine in the injured ischemic rat brain. Neuroscience 168: 219225.
30. Yang HJ, Ishizaki I, Sanada N, Zaima N, Sugiura Y, et al. (2010) Detection of
characteristic distributions of phospholipid head groups and fatty acids on
neurite surface by time-of-flight secondary ion mass spectrometry. Med Mol
Morphol 43: 158164.
31. Yang HJ, Sugiura Y, Ikegami K, Konishi Y, Setou M (2011 ) Axonal gradient of
arachidonic acid-containing phosphatidylcholine and its dependence on actin
dynamics. J Biol Chem: Dec 29. [Epub ahead of print].
32. Back M, Bu DX, Branstrom R, Sheikine Y, Yan ZQ, et al. (2005) Leukotriene
B4 signaling through NF-kappaB-dependent BLT1 receptors on vascular
smooth muscle cells in atherosclerosis and intimal hyperplasia. Proc Natl Acad
Sci U S A 102: 17501756.
33. Riccioni G, Zanasi A, Vitulano N, Mancini B, DOrazio N (2009) Leukotrienes
in atherosclerosis: new target insights and future therapy perspectives. Mediators
Inflamm 2009: 737282.
34. Zhao L, Moos MP, Grabner R, Pedrono F, Fan J, et al. (2004) The 5lipoxygenase pathway promotes pathogenesis of hyperlipidemia-dependent
aortic aneurysm. Nat Med 10: 966973.
35. Yokote K, Morisaki N, Zenibayashi M, Ueda S, Kanzaki T, et al. (1993) The
phospholipase-A2 reaction leads to increased monocyte adhesion of endothelial
cells via the expression of adhesion molecules. Eur J Biochem 217: 723729.
36. Sakai M, Miyazaki A, Hakamata H, Sato Y, Matsumura T, et al. (1996)
Lysophosphatidylcholine potentiates the mitogenic activity of modified LDL for
human monocyte-derived macrophages. Arterioscler Thromb Vasc Biol 16:
600605.

February 2013 | Volume 8 | Issue 2 | e57398